Photophyiscal and Biological Implications of the Fluorophore Polymer Ratio.

Abstract

Multivalent polymers can be used as gene delivery vectors, diagnostics, and therapeutics when they are conjugated with small molecules. The random nature of attaching small molecule ligands to a polymer leads to a large mixture of different ratios that can be approximated by one or more Poisson distributions. The variety of ligand to polymer ratios leads to a large difference in hydrophobic character, which can alter biological properties. The hydrophobicity ratio has been known to effect the polymer’s behavior in solution and cell. The exact effect of this ratio was determined using precise ratio materials isolated. The precise ratio of fluorophores on a polymer is specifically of interest because of the environmental dependence on fluorophore properties. The precise ratios of a fluorophore on a polymer also provides a model system for understanding how changes in hydrophobicity alter a multivalent polymer’s biological behavior. Precise dye-polymer ratios of fluorescein and TAMRA were obtained on an acetylated G5 PAMAM dendrimer by using semi-prep rp-HPLC. The fluorescein-G5 conjugates were obtained by cycloaddition of an azide functionalized fluorescein molecule with a precise ratio of cyclooctyne ligands on a G5 PAMAM dendrimer. The TAMRA-G5 PAMAM conjugates were obtained by direct conjugation and isolation. Both sets of materials were fully characterized. Amine-terminated G3 PAMAM dendrimers with precise ratios of TAMRA were also obtained and fully characterized. Similar absorption and emission spectra were obtained compared to the neutral TAMRA-G5 materials. Precise ratios of TAMRA on amine-terminated G5 PAMAM dendrimers were studied in HEK 293A cells. Dendrimers with 2-4 TAMRA were found to have greater uptake than dendrimers with a single TAMRA. FLIM showed different ratios of TAMRA alone was sufficient to change the fluorescence lifetime of the material observed inside cells. Multiple distributions of small molecules to the polymer will occur when conjugating a fluorophore and a targeting ligand or drug. Using amine terminated precise ratio TAMRA-G5 PAMAM material, controlling a second distribution of small molecules on a polymer was investigated. Multiple strategies are discussed along with the advantages and disadvantages of each strategy.